G. Yu. Lomakina, Yu. A. Modestova, and N. N. Ugarova*

Received January 16, 2015
Theoretical aspects of the adenosine triphosphate bioluminescence assay
based on the use of the firefly luciferin–luciferase system are
considered, as well as its application for assessing cell viability in
microbiology, sanitation, medicine, and ecology. Various approaches for
the analysis of individual or mixed cultures of microorganisms are
presented, and capabilities of the method for investigation of
biological processes in live cells including necrosis, apoptosis, as
well as for investigation of the dynamics of metabolism are described.
KEY WORDS: bioluminescence, firefly luciferase, ATP,
intracellular ATP, cell lysis, extracellular ATP, lyophilized vaccines

DOI: 10.1134/S0006297915060061

GENERAL CHARACTERISTICS OF BIOLUMINESCENCE ASSAY OF INTRACELLULAR
ATP

ATP as an indicator of cell viability. Adenosine triphosphate
(systematic name 9-β-D-ribofuranosyl adenine-5′-triphosphate
or 9-β-D-ribofuranosyl-6-amino-purine-5′-triphosphate)
− a nucleotide, an adenosine triphosphate ester that is a
derivative of adenine and ribose (ATP) − is the main energy
carrier in cells of all living organisms (mammals, microorganisms,
plants, etc.) [1]. Cleavage of one or two phosphate
groups that occurs during ATP hydrolysis is accompanied by the release
of energy. In cells, ATP transfers energy to other molecules upon
hydrolysis to its low-energy analogs (ADP and/or AMP), which, in turn,
acquire energy by adding phosphate groups and transforming into ATP.
Intracellular ATP content is the main indicator of cell viability. Upon
cell death, ATP synthesis is the first to be arrested, while its
hydrolysis can continue for some time, hence, the intracellular ATP
content drops sharply to zero value. The ATP content in viable cells of
microorganisms is quite high – it ranges from 500 to
10,000 µg per g of dry biomass [2] or
from 10–19 to 10–15 mol of ATP
per cell. It can vary depending on the type and size of cells and their
energy state. Cellular response to any stress involves change in the
intracellular ATP content. The intracellular ATP concentration can also
vary depending on cultivation conditions, but the physiological changes
that do not lead to the cell death do not exceed 10-50-fold. On the
example of individual strains of microorganisms, it was shown that the
intracellular ATP content for a cell suspension under standard
conditions is proportional to the concentration of cells in the
suspension [3]. Hence, it is possible to estimate
the number of viable cells in a sample by measuring the intracellular
ATP concentration.

Determination of ATP concentration using bioluminescence assay.
There are various methods to determine ATP concentration: enzymatic
methods with spectrophotometric detection, radioactive and
chromatographic methods, and others. The bioluminescence ATP assay is
the most sensitive, rapid, and selective. As far back as in the 1940s,
it was shown that ATP is a required component in the reaction catalyzed
by the firefly luciferase enzyme [4]. The overall
reaction that proceeds in the luciferin–luciferase system of
fireflies is described by the following scheme:

According to this scheme, an organic substrate (luciferin) is rapidly
oxidized in the presence of ATP and magnesium ions by air oxygen to
oxyluciferin with simultaneous formation of pyrophosphate (PPi) and
AMP. Initially the oxyluciferin is formed in an electronically excited
state, and a light quantum is emitted during the transfer of the
product to the ground state. The advantages of firefly luciferase
include its absolute specificity towards ATP and high emission quantum
yield (~0.5 – the highest among known bioluminescence systems)
[5].

Luminometers – specialized instruments that were first introduced
to the market in 1970s – are used for bioluminescence
measurements. The first luminometers were based on the technology
developed for detection of radioactive emission – liquid
scintillation counters [6]. Eventually another
approach was developed, a photon counter in combination with a
high-efficiency photomultiplier tube (PMT) is used in modern
instruments for detection of luminescence signal. It must be noted that
the signal recorded by a luminometer is proportional but not equal to
the number of photons emitted by the sample. The signal is presented in
relative luminescence units (RLU). Its absolute value is determined by
the parameters of the particular instrument and can differ even for
luminometers of the same model [7]. The light
intensity recorded in RLU is proportional to the ATP concentration in
the ATP linear range from 10 fM to 1 µM. There are a great
variety of modern luminometers that differ in a number of parameters:
sensitivity, type (tube luminometers and plate readers), size and
design complexity (stationary and portable), availability of injectors
or lack of them (i.e. intended for recording stationary (glow-type) or
pulsed (flash) luminescence), etc. The portable luminometer LUM-1
– high-sensitivity photon counter capable of recording
luminescence intensity in the range from 10 to 800 thousand pulses/s
and with built-in computer interface – is manufactured in Russia
(Fig. 1). The LUM-1 luminometer demonstrates
the same sensitivity and operation stability as instruments of the same
class from Berthold Detection Systems GmbH (Germany).

Fig. 1. LUM-1 portable luminometer (Lumtek,
Russia).

Universal instruments capable of operating in multiple modes such as
recording luminescence, fluorescence, and absorption are also available
commercially. Accuracy of results is determined primarily by the
selection of a suitable instrument for conducting analysis.

To unify the methods of analysis, dozens of foreign companies offer
reagent kits for bioluminescence ATP assay, the so-called ATP reagents.
The ATP reagents comprise lyophilized mixtures containing all
components required for the luciferase reaction to proceed (luciferase,
luciferin, magnesium salt, buffer component, and stabilizers) except
for ATP. The lyophilized ATP reagents are reconstituted prior to use by
adding a special solution for the ATP reagent reconstitution. The ATP
reagents based on Russian luciferase from Luciola mingrelica
fireflies have been developed [8, 9]. The reagents include a mutant recombinant
luciferase that is significantly more active and thermostable than
native luciferase [10, 11].
The sensitivity of the modern bioluminescence test systems allows
detecting attomolar quantities of ATP in a sample and revealing the
presence of single cells of microorganisms [12].
For quantitative determination of intracellular ATP, the cells are
lysed to release the ATP, and the extract is introduced into the
measuring cell, then the ATP reagent is added and the luminescence
intensity is measured. To calculate accurate ATP concentration in the
sample, a control is usually used that employs a standard ATP solution
(ATP control) containing a known amount of ATP. The lyophilized ATP
control is usually supplied together with the ATP reagent. The ATP
control is reconstituted with the same extractant used for the cell
lysis in the sample, and then the luminescence intensity is measured.
The ATP concentration in the analyzed sample can be calculated by
comparison with the standard.

The construction of graduation dependence between concentration of
intracellular ATP and concentration of cells in a sample is an
important step in quantitative determination of viability of microbial
cells. Data of the standard method used for evaluation of the viability
of microbial cells – serial dilution – are used for this
purpose. The number of viable cells (CFU/ml) is determined using the
standard method, and the ATP content in 1 ml of the sample is
determined using the bioluminescence method. Linear correlation
dependencies (y = a + bx) are obtained as a result:

[ATP], mol/ml = a + b·(CFU/ml),

which are then used for determination of the (CFU/ml) value based on the
value of [ATP], mol/ml. Good linear correlation between ATP
concentration and CFU/ml is observed for individual cultures in the
range from 103 to 108 CFU/ml (Fig. 2).

The bioluminescence method allows rapid and quantitative determination
of ATP amount. For this reason, it is used in various areas of
molecular biology, sanitation, medicine, clinical diagnostics, food
industry, ecology, and others. The possibilities and features of the
use of the bioluminescence method for determination of the viability of
microorganisms are presented below.

DETERMINATION OF INTRACELLULAR ATP CONTENT IN INDIVIDUAL CULTURES
OF MICROORGANISMS

Intracellular ATP is inaccessible to luciferase because the enzyme does
not penetrate inside cells. Reagents that increase cell membrane
permeability or disrupt it are used for the extraction of intracellular
ATP. The type of the analyzed cells and the needs of the particular
analysis primarily define the selection of the lysing agent.
Extractants used for ATP assay must: (i) rapidly extract ATP; (ii)
inactivate intracellular enzymes participating in interconversion of
adenine nucleotides within the cell; (iii) maintain the initial ATP
level of the intracellular ATP during extraction, storage, and
analysis; (iv) not exhibit an inhibitory effect on the luciferase
reaction during the ATP measurement.

Various types of compounds can be used for the ATP extraction:
detergents and their mixtures, strong acids (i.e. perchloric and
trichloroacetic), organic solvents (butanol, chloroform, dimethyl
sulfoxide, etc.). Their application simplifies the experiment and
allows conducting differential analysis based on different permeability
and strength of the cell wall of different organisms in the presence of
the extractants. Often a combination of chemical and physical methods
for cell disruption is used: disintegration of cells by short-term
boiling, sonication [13], or direct current [14].

Efficiency of ATP extraction from microorganisms depends mostly
on the nature of the cell wall. Cytoplasmic membranes of microorganisms
have similar structure and are easily disrupted with weak acid
solutions or surfactants. The cell wall structures of Gram-positive and
Gram-negative bacteria and of yeasts differ significantly, and these
features must be considered upon selection of the lytic agent. For
example, it was shown in [15], when the efficiency
of different types of extractants was compared, that the maximum
sensitivity of the bioluminescence analysis of intracellular ATP of
Gram-positive bacteria and yeasts was observed using 0.1% solution of
the detergent cetyltrimethylammonium bromide (CTAB). It was one order
of magnitude higher in comparison with the 2.5% trichloroacetic acid
(TCA) and 90% dimethyl sulfoxide (DMSO). This is mainly related to the
fact that CTAB disrupts cells rapidly and effectively, while it
inhibits luciferase significantly less in comparison with TCA and DMSO.
However, CTAB was not applicable for the analysis of highly
concentrated suspensions of Gram-negative microorganisms such as E.
coli and Pseudomonas sp. It was not possible to use CTAB
because noticeable deviations from linearity of the dependence of the
ATP concentration on the cell concentration were observed when it was
used. This is due to the fact that Gram-negative bacteria, in contrast
to Gram-positive bacteria and yeasts, have significantly stronger cell
wall, and, hence, stronger chemical agents are required to disrupt it.
Unlike CTAB, DMSO was universal; it disrupted the cell walls of all
investigated microorganisms instantly and inactivated all intracellular
enzymes. The extraction time did not exceed 1 min. The resulting
extracts were stable at room temperature for several hours, and it was
possible to store them at 4°C for several days and even weeks,
while the extracts obtained using CTAB were unstable during storage.
Nevertheless, the use of detergents including CTAB for ATP extraction
is the most popular method, and it is used in analysis very often. For
example, cells were treated with 5 mM CTAB for 2 min for ATP
extraction when a disposable sensor for assessing microbiological
cleanness of a surface was developed [16].

The cationic detergent benzalkonium chloride (BAC), which compared well
in efficiency with TCA, was used in [17] for
analysis of intracellular ATP in 17 yeast strains (with suspension
concentration of 105 CFU/ml) and 37 strains of Gram-positive
and Gram-negative microorganisms (with concentration of 107
CFU/ml). It was shown that the average degree of ATP extraction by 0.2%
BAC solution from Gram-positive bacteria was above 99.4%, and from
yeast cells – above 97%. The extraction efficiency from
Gram-negative bacteria was significantly less (~81%) in comparison with
the TCA extraction. Especially low extraction was observed for
Escherichia, Proteus, and Serratia microorganisms
(74-80%). Increase in BAC concentration to 0.5% resulted in inhibition
of the luciferase activity and significant increase in the
bioluminescence signal decay rate from 5 to 50%/min. One hundred
percent extraction of ATP from fungal spores was achieved upon
treatment of the sample with 90% DMSO in TAE buffer, pH 7.75, for
1 min at 100°C [18]. For comparison, the
efficiency of ATP extraction with boiling TAE buffer without DMSO (it
was no more than 37.7%) and with 0.1-3% TCA solution (it was up to
23.3%) was assessed.

Special extraction conditions are required for analysis of mycobacteria,
which differ from the other Gram-positive bacteria by unusual structure
of their cell wall. In mycobacteria, the dense layer of peptidoglycan
(the feature of Gram-positive bacteria) in the cells of this type of
bacteria is strengthened by mycolic acid, mycolates, and
arabinogalactan; their surface is hydrophobic due to the presence of
lipids [19]. Dodecyltrimethylammonium bromide (2%)
in Tris-EDTA buffer (pH 7.75) (100°C, 1 min) in the
presence of α-cyclodextrin was among the optimal extractants [20]. An approach combining mechanical cell disruption
(bead beating) and enzymatic action of lysozyme (Bactozyme preparation)
[21] was also suggested. It was shown in [22] that the use of such extractants as TCA,
perchloric acid (PCA), and ethylene glycol was undesirable for ATP
extraction from cells with high protein content because their presence
caused protein denaturation and partial ATP coprecipitation. Phenol
saturated with Tris-EDTA is recommended for this case; this approach
increases the ATP yield 14.5-fold in comparison with 5% TCA and
1000-fold in comparison with ethylene glycol.

Various procedures can be employed to account for the effect of anionic,
cationic, and amphoteric detergents on the luciferase activity. One of
the classic methods involves the use of various cyclodextrins [7]. For example, 7.5 mM β-cyclodextrin was
used to decrease the inhibiting effect of 5 mM CTAB [16]. Liposomes were used to bind BAC following the
ATP extraction from bacterial cells [23]. The
addition of diethylaminoethyl dextran increased the luciferase activity
in the presence of TCA and Triton X-100 [24].
Genetic engineering approaches are also used. For example, a double
mutant of luciferase resistant to BAC was used in [17] to decrease the inhibiting effect of this
extractant on the luciferin–luciferase reaction.

Intracellular ATP level is different in cells of different type
and depends on many factors. The review [25]
devoted to practical aspects of the bioluminescence assay application
presents data from different sources on the ATP content in cells of
bacteria and yeasts, in the spores of both bacteria and fungi, and in
actinomycetes. It is stated that the ATP content in bacterial cells can
differ by 4-5 orders of magnitude. For example, the ATP content of
Gram-negative bacteria during the stationary growth phase was within
the narrow range (~10–18 mol/CFU), while the ATP
content of Gram-positive bacteria varied in a wide range from
0.4⋅10–18 to
16⋅10–18 mol/CFU, but on average it was one
order of magnitude higher as compared with the Gram-negative bacteria
[17]. The intracellular ATP content of different
strains of yeast cells was in the range
(0.7-54)⋅10–16 mol/CFU. The ATP content in
the cell can be related with its size [26] and
volume [27]. The intracellular ATP concentration
is generally rather high; on average, it is about 1-10 mM.
According to [28], the ATP concentration in the
E. coli cell is no less than 3 mM and independent of the
cell propagation rate in the mid-log growth phase. In 2014, a group of
scientists was able to determine an absolute content of ATP in
individual E. coli cells using the genetically encoded
fluorescent ATP indicator QUEEN [29]. It was found
that the ATP content within a cell population has a positively
asymmetric distribution; average ATP concentration within an individual
cell was 1.54 ± 1.22 mM. The averaged data obtained using
fluorescence (QUEEN) and bioluminescence analysis were in full
agreement with each other. Moreover, using five different species of
marine bacteria, it was shown that there is a good correlation between
the ATP and carbon content in the cell [30].

Evaluation of metabolic activity of cells based on intracellular ATP
content. An increasing number of researchers use the
bioluminescence method as a powerful tool for evaluation of the
metabolic activity of cells. When using the bioluminescence assay, the
detection limit of bacterial cells may essentially depend on the phase
of their life cycle. On the example of E. coli and S. aureus
cells [31], it was shown that the level of
intracellular ATP changes depending on the growth phase of the
microorganism (lag-phase, log-phase, stationary phase, and cell death).
Metabolic activity changes in S. cerevisiae during growth and at
different temperatures were studied in [32]. Three
sub-populations of the yeast that respond differently to elevated
temperature were distinguished: viable cells, dead cells, and viable
but nonculturable cells (hence undetectable by the plating method). The
ATP level in the cell can increase with increasing temperature because
of activation of metabolic paths essential for the cell to withstand
this stress.

Bioluminescence analysis was used in [33] to study
the effect of cell adhesion to a glass surface on metabolic activity
and, respectively, on the level of intracellular ATP. It was
established that the intracellular ATP content increased 2-5-fold
during adhesion of E. coli and B. brevis cells to the
glass surface in comparison with the level characteristic of the cells
in suspension. Data obtained in [34] indicate that
the level of metabolic activity in the cell depends on the properties
of the adhering surface: for a number of cells, adhesion to a
hydrophobic surface resulted in two-fold increase in the intracellular
ATP content in comparison with the same cells adhered on a hydrophilic
surface.

Kinetic analysis of change in intracellular ATP content as well as
analysis of ATP/ADP ratio allows investigating various cell pathologies
including mechanisms of cell death – necrosis and apoptosis. As
long as the cells are alive, they tend to maintain normal ATP level,
whereas the level of intracellular ATP decreases to zero upon cell
death. And yet the rate of ATP hydrolysis in cells is different.
Mechanical damage of cytoplasmic and intracellular membranes occurs
under the action of biological, physical, or chemical factors during
necrosis, which results in disruption of organelles, release of
lysosomal enzymes, and release of the cytoplasmic content into the
extracellular space. Mechanisms of necrosis do not require energy,
unlike apoptosis, during which the cell attempts not only to maintain
but to increase ATP level for several hours to supply energy for the
process via glycolysis or synthesis of mitochondrial ATP. The type of
cell death – via apoptosis or necrosis – is determined
mainly by the intracellular concentrations of NADH and ATP. The
bioluminescence method was used for determination of intracellular ATP
when investigating mechanisms of cell death of macrophages under the
action of different strains of M. tuberculosis and showed that
ATP synthesis increased sharply in the cells infected by the virulent
strain H37Rv, unlike in cells in the presence of nonvirulent H37Ra
strain [35].

Bioluminescence methods for evaluation of cytotoxicity of various
preparations against bacterial cells. Good agreement was obtained
between the bioluminescence method and laser nephelometry when
antibacterial properties of a series of cyclodextrin complexes were
evaluated [36]. To assess the efficiency of new
chemical disinfectants, standard techniques developed by the European
Committee for Standardization, EN 1276 and EN 13727 in particular, are
used. These standards describe the analysis of bacterial suspensions.
They prescribe the following steps in the measurement: a biocide
solution is mixed with the bacterial suspension in the presence of
organic matter (bovine serum albumin, for example) that mimics
conditions under which the test solution is used in practice. Following
a prescribed time period, the activity of the effector is inhibited and
the viability of the microorganisms is determined using the colony
counting technique on nutrient medium. It was shown in [37] that these techniques could be significantly
simplified and optimized if the colony counting technique was replaced
by semiautomatic bioluminescence assay for bacteria viability. The
bioluminescence method was also used for cytotoxicity screening of
pharmaceutical products against Leishmania promastigotes (a type
of parasitic protozoans) [38]. Bioluminescence was
also used in [39] for quantitative assessment of
proliferation of another protozoan – Perkinsus marinus,
which is an oyster parasite. The efficiency of oral antiseptics was
assessed in [40] using bioluminescence analysis.
The bioluminescence method for assessing the effectiveness of
mouthwashes against biofilms grown in vitro using mouth
microflora was validated in [41].

Biological fluids can be analyzed for ATP content without pretreatment
of the sample because the turbidity and coloring of the liquid do not
significantly interfere with the analysis. For example, the effect of
radiation and xenobiotics on intracellular ATP content was investigated
in [42] not only for blood cells (erythrocytes and
neutrophils), but also for the whole blood, which was diluted 10-fold
with physiological solution prior to analysis; DMSO was used as the
extractant. The dynamics of ATP content and its absolute values
indicated metabolic changes. The observed increase in the ATP level in
whole blood and in neutrophils was statistically significant. An
interesting approach for application of bioluminescence assay was
suggested in [43]. The authors used a plating
method for detection of bacterial cells and spores, which was modified
as follows: the sample was plated onto a filter surface, which was
placed on the surface of solidified nutrient medium and incubated for a
certain time that depended on the growth rate of the cells of this
type. Next the filter was transferred into a special device where the
filter surface was treated to remove extracellular ATP, and the
intracellular ATP was extracted from the colonies grown on the filter
that were still invisible to the naked eye. Then the filter surface was
treated with the ATP reagent, and luminescence was recorded with a
sensitive CCD camera. This shortened the time required for the cell
growth, and it was possible to count the colonies before they became
visible to the naked eye.

DETERMINATION OF INTRACELLULAR ATP IN A MIXED CULTURE OF
MICROORGANISMS

The method for determination of intracellular ATP can be applied not
only for analysis of individual cultures, but also of their mixtures,
and it can be successfully used instead of time-consuming
microbiological analysis in cases when knowledge of total microbial
counts is required. In work [26], bioluminescence
ATP assay was used for analysis of sterility of indoor facilities.
Plating onto solid TSA medium followed by colony counting was used for
detection of culturable microorganisms, and also the average ATP
content in a live cell – ATP/CFU ratio – was determined. In
the case of low ATP/CFU ratio, it can be assumed that predominately
Gram-negative microorganisms and spores are present in the sample,
while if the ratio is high – Gram-positive bacteria and yeasts.
It was found that a large number of samples did not form any colonies
on the nutrient medium, and yet a high level of intracellular ATP was
recorded. This suggested the presence of viable but nonculturable
microorganisms, which was later confirmed by DNA analysis. Hence,
bioluminescence ATP assay can be used for express detection of
nonculturable or slowly growing microorganisms. It must be mentioned
that despite a wide variety of microorganisms in nature, only a limited
number can be cultivated under laboratory conditions. Data on the
efficiency of the cultivation characteristic of bacteria in different
habitats are presented in review [44]. The least
effective cultivation is usually observed for bacteria living in
environments with low nutrient levels (below 1%), and the highest for
bacteria living in nutrient-rich habitats (up to 58%).

Determination of total bacterial count in air (TBC, CFU/m3),
1 m3 of which can contain up to 106 cells of
aerobic pathogenic and nonpathogenic microorganisms (fungi,
bacteria, spores, and others), is very important for analysis of
sanitary quality of air in facilities with different air quality
requirements (according to ISO 14644-1-99), including sterile and clean
rooms (0-10 CFU/m3) in health care facilities, industrial
premises, and offices in food and microbiological industries. The
commonly used microbiological method requires no less than 24 h. A
semiquantitative method for evaluation of TBC in air in rooms with low
concentrations of microorganisms was suggested in [45]. Air was pumped (optimal air flow 200 liters/min)
through a wet sterile adsorbing surface to capture aerobic
microorganisms, and the samples were incubated in nutrient medium for
3-6 h to the beginning of the logarithmic growth phase. Both
Tryptic Soy Broth (Difco, USA) and nutrient broth (State Research
Center for Applied Microbiology and Biotechnology, Russia) were found
to be suitable. Good agreement between the bioluminescence assay and
the microbiological plating method was obtained: 3-h incubation of the
sample at 37°C revealed 50-100 CFU/m3 (cleanness grade
6-7), and 5-h – 5-50 CFU/m3 (cleanness grade 5,
especially clean facilities). If the total count of microorganisms was
≥500 CFU/m3 (grade 9 and above), the result of analysis
was obtained within 5 min without prior incubation.

A system for automatic investigation of bioaerosols was described in [46]; it comprised an atomizer for preparation of
bioaerosols from suspension of bacterial cells, an air ionizer, and a
measuring unit of complex design in which cell lysis occurred under the
action of ionized air followed by bioluminescence ATP assay. The
authors automated the experiment, decreased the analysis time to
5 min, and detected bacterial cells (E. coli and S.
epidermidis) at concentrations from 173 to 541 CFU/m3
without enrichment.

Adhesion of bacterial cells to surfaces of various nature (glass, metal,
plastic, etc.) results in formation of biofilms, which create
significant problems in both medicine and industry. Bioluminescence
analysis allows the investigation of the formation of communities of
microorganism comprising biofilms, their existence, survival, and
distraction under the action of different effectors [41, 47-50].

Determination of cells of individual microorganism in the presence of
accompanying microflora is a rather difficult task. Different
approaches are used for this purpose. The traditional way to
selectively isolate cells of a target strain is cultivation of the
sample on selective nutrient medium, which supports preferable growth
of the strain of interest. For example, milk samples were incubated in
selective nutrient medium for 6 h for determination of
coli-form organisms in food (raw milk and ice cream) and rinses,
which allowed the authors to detect 10 CFU/ml in a sample [51]. The authors in [52] used an
immunomagnetic separation technique for specific capture of E.
coli O157:H7 by antibodies attached to magnetic particles. The cell
content was then determined from the level of intracellular ATP. The
reached limit of detection was 102 CFU/ml.

The use of lytic bacteriophage for rapid and specific lysis of target
cells allows reliable detection of 104 CFU/ml in the
presence of nonspecific microflora. Concentration of the initial cell
suspension prior to detection improves the analytical characteristics
of the method by one order of magnitude. In such manner, positively
charged nanoaluminum fiber-based filters with pore size of
2 µm with adsorbed T4 bacteriophage allowed quantitative
determination of E. coli cells in the presence of 60-fold excess
of S. typhimurium [53]. A photodynamic
method for selective disruption of Gram-negative and Gram-positive
bacteria, using strains of E. coli O157:H7 and L.
monocytogenes as an example, as well as for specific detection of
yeast cells in mixture with E. coli was used in [54, 55].

Total ATP content in a sample represents a convenient indicator
for estimation of hygienic conditions by characterizing residual
content of live and disrupted bacterial or somatic cells. A large
number of studies have been devoted to express analysis of rinses from
industrial surfaces, dishes, equipment, food products, etc. All cells
provide ATP, both microbial and non-microbial (somatic), as well as
cellular matter of organic nature, for example, food residues.
Treatment of cells with disinfecting agents results in their
destruction and release of ATP. Hence, this method can be employed for
evaluation of the efficiency of disinfecting agents and quality of
sanitation of an object. Special instrumentation has been designed and
is widely used for this purpose that allows primary screening of
hygienic condition of facilities on-site and in real time. Numerous
companies (Biotrace from New Zealand; HY-LiTETM, Merck,
Hygiena International, and Berthold Detection Systems GmbH from
Germany; New Horizons Diagnostic Corp. and Pierce from USA; BioThema
from Sweden; Kikkoman Corp. from Japan; Roche Diagnostics Ltd. from
Switzerland; Lumtek from Russia, and others) offer various kits for
analysis that contain reagents, disposables, and luminometers. The
Hygiena International, for example, offers single-use devices for
sampling and measuring of total ATP content in combination with a
portable luminometer. The method was tested in food industry [56] for monitoring of microbial contamination on
hands and household surfaces [57], for assessing
hygienic conditions of technological surfaces in food-handling
facilities [58], and in hospitals [59]. The method is quite effective for real-time
monitoring of the cleanliness of medical equipment, such as endoscopes,
in the process of their disinfection, which allows prevention of
cross-infection among patients [60].

However, it has been mentioned in a number of publications that low
correlation was observed between the results of control microbiological
tests and bioluminescence assay while the method was validated in
industry and large amounts of data have been statistically processed
[56]. Various factors could cause false
negative results in determination of intracellular ATP: incomplete
cell disruption or incomplete extraction, low rate of extraction,
decrease in intracellular ATP concentration due to its hydrolysis by
ATPases during extraction, low stability of the bioluminescence signal,
ATP disruption during storage of extract, etc. In the development of an
ATP extraction technique for a particular sample, it is very important
to make sure that the method yields correct data on intracellular ATP
content in the sample. Observed false-positive results are
usually related to a high level of the extracellular ATP, which occurs
when cells are already disrupted but not removed. In this case, it is
necessary to differentiate total ATP content into extracellular
(disrupted cells) and intracellular (live cells) ATP.

Determination of extracellular ATP content. The amount of
extracellular or so-called “free” ATP in a cell suspension
is usually one or two orders of magnitude lower than the intracellular
ATP. For example, the extracellular ATP content in a culture of
metabolically active E. coli and Salmonella cells
constituted no more than 5% of the total ATP pool [61], and it was defined by the growth phase of the
cells. The level of extracellular ATP increased during the logarithmic
phase and decreased during the stationary phase. The sole exception was
the Acinetobacter junii AJ4970 cells, for which the ratio of
extracellular to intracellular ATP was found to be above 0.5. It was
shown that the bacterial cells rapidly consume extra free ATP with rate
of approximately 5 µmol/h. According to [61], an artificial increase in ATP concentration in
nutrient medium resulted in better survival of cells during prolonged
incubation (7 days).

The extracellular ATP level can increase significantly under external
stresses due to escape of intracellular ATP into the environment. The
extracellular ATP content can be indicative of the number of destroyed
cells upon, for example, freezing, in the process of lyophilization,
under unfavorable storage conditions, etc. Live and disrupted cells can
be distinguished by the ratio of extra- and intracellular ATP. For
example, a method is described in [62] for
quantitative determination of bacteria sensitivity to nisin – a
broad spectrum antibiotic affecting Gram-positive bacteria that is
widely used as a preservative. Under the action of nisin, cells release
ATP, ADP, and AMP into the environment. Using the enhanced sensitivity
of Lactococcus cremoris cells to this antibiotic, it was shown
that the nisin concentration could be determined with the
bioluminescence method from the amount of the extracellular ATP.

As shown in [34], the amount of extracellular ATP
secreted by a series of Gram-positive and Gram-negative bacteria
depended on the nature of the surface in contact with the
microorganisms: contact with a surface unfavorable for cells stimulated
the level of ATP release, and the level of extracellular ATP for
Gram-negative bacteria was on average higher than for Gram-positive
bacteria.

Importance of extracellular ATP removal. Hence, to correctly
determine intracellular ATP, it is necessary to first remove
extracellular ATP, followed by extraction of the intracellular ATP. Two
main methods are used for removal of extracellular ATP: a) filtration
of cell suspension through a membrane filter during which the
extracellular ATP is removed together with the filtrate [63]; b) enzymatic hydrolysis of the extracellular ATP
[64]. Selection of one method or the other is
based on peculiarities of the analyzed suspension of microorganisms.
Filtration is used in cases when the determined concentrations of
intracellular ATP are less than 1 pM (at low concentrations of
cells, which is, for example, lower than 102 CFU/ml for
Gram-negative microorganisms), which at the same time results in
concentration of cells on the filter and increase in the measured ATP
concentration. The enzymatic hydrolysis of ATP is preferable when the
cell concentrations are sufficiently high (above 104
CFU/ml).

Special 300-µl cuvettes with filtering bottom having pore size of
0.45 µm (Filtravette™; New Horizons Diagnostic Corp.,
USA) were used in [65] for determination of total
microbiological contamination of food products. The removal of
extracellular ATP, concentration of bacterial cells, ATP extraction,
and measuring of its content using the bioluminescence method is
conducted sequentially in the same cuvette. In the case when the sample
contains more than 1000 CFU/ml (for E. coli), the analysis time
is no more than 5-10 min. Preincubation of the sample for 6 h in
nutrient medium to increase metabolic status of the cells and their
propagation allowed detection of 1 CFU/ml in drinking water [66]. The same approach was used in [67] for control of sterility in health care
facilities. The lack of bioluminescence signal following 6 h of
incubation of rinses from different surfaces in nutrient medium
indicated sterility of the object. In the case when a positive
bioluminescence signal was observed after 6 h of incubation, the
microbial contamination of the sample was 1-10 CFU/100 cm2.
Microbial contamination above 100 CFU/100 cm2 was detected
without incubation. The correlation coefficient between the ATP and CFU
was 0.8-0.95. The problem of high content of non-bacterial ATP also
presents challenges for analysis of purity of soya milk. The possible
sources of non-bacterial ATP in soya milk are fibers and proteins in
its composition [68]. Pretreatment of such a
sample with a mixture of nonionic detergents [68]
and removal of non-target ATP allowed using the bioluminescence method
for sterility testing of such products.

Total bacterial counts or quantity of mesophilic aerobic and facultative
anaerobic microorganisms (QMAFAnM) represent one of the main indicators
of the sanitary state of raw milk, which determines ways for further
processing of the milk and affects the final price of the product. The
generally recognized microbiological method according to State Standard
GOST 9225-84 (Russia) allows determination of QMAFAnM in milk following
72 h incubation of plated samples; hence, express methods for
evaluation of product quality, including the bioluminescence ATP assay
with total analysis time of 20 min and detection limit 1000
CFU/ml, are of particular interest. For this purpose, a milk sample is
incubated with a mixture of detergents and protease to destroy somatic
cells and protein micelles in the milk. Next, the non-microbial ATP is
removed by filtration through a bacterial filter, the bacteria are
disrupted with DMSO, and the concentration of intracellular ATP is
measured [69] (Fig. 3).
By way of illustration, if the concentration of microbial ATP is less
than 0.6 pmol/ml, the sample contains up to
5·104 CFU/ml, and if above 70 pmol/ml –
more than 4·106 CFU/ml.

Fig. 3. Scheme of bioluminescence determination of
intracellular ATP in a biological sample.

A combined method for determination of spore contamination of food
products was suggested in [70]. In the first step
of the analysis, the authors used bioluminescence ATP assay in the
sample: the sample was concentrated in a Filtravette™, somatic
cells were disrupted by treatment with a mild lytic agent, and both
somatic and free ATP was removed. To increase the intracellular ATP
concentration, germination of spores was activated in nutrient medium.
If the results were positive, indicating the presence of bacterial
contamination, the samples were further analyzed for the presence of
sporulation gene spo0A with real time PCR using SYBR Green
dye.

Sensitivity of bioluminescence ATP assay can be increased not
only by concentration of cells on bacterial filters or their enrichment
in suitable nutrient medium. The ATP content in a cell is related to
the content of ADP and AMP, which form a system of adenine nucleotides
in the cell. The reversible equilibrium reaction catalyzed by adenylate
kinase enzyme plays an important role in maintaining equilibrium
between them in the system of adenine nucleotides in the cell:
ATP + AMP = 2 ADP.

The authors in [71] suggested destroying single
live cells collected on a filter following removal of extracellular and
somatic ATP and using the hybrid protein adenylate
kinase–polyphosphate kinase to convert the endogenous ATP in the
presence of AMP and polyphosphate into double the amount of ADP and
again to ATP. The method allowed detection of 75 CFU of S.
aureus in 0.5 ml of milk. The sensitivity of the analysis
increases 10,000-fold in the process and allows detecting
10–18 M ATP.

CELL VIABILITY ASSESSMENT IN VACCINES BASED ON LYOPHILIZED
MYCOBACTERIA CELLS

An important problem of modern microbiology is development and
optimization of methods for extended storage of cultures of
microorganisms, specifically lyophilization. Survival of cells
following lyophilization depends on a number of parameters such as
composition of the nutrient medium, initial cell concentration,
availability and type of cryoprotectors, mode of lyophilization, and
method of subsequent rehydration [72]. A method
for rapid assessment of viability of microbial cells is required for
optimization of these parameters. Traditionally plating is the method
of choice, but bioluminescence analysis, especially suitable for
viability testing of slowly growing organisms, can serve this function.
A number of publications describe application of bioluminescence
analysis for assessment the viability of lyophilized BCG vaccines [73, 74].

The viability of microbial cells in vaccines based on lyophilized cells
of mycobacteria is a main indicator of their quality. When the standard
microbiological method is used, the suspension of rehydrated cells is
inoculated onto solid nutrient medium, and the number of formed
colonies is counted following incubation. Mycobacteria are slow-growing
organisms, and the incubation period could last up to five weeks.
Moreover, the standard method is very variable and often hard to
reproduce because mycobacterial cells are prone to aggregation. All
this complicates the production and testing of vaccine preparations.
The bioluminescence method provides high sensitivity, speed, accuracy,
and repeatability of determination of viable cells in vaccine. This is
the reason why the first studies on application of bioluminescence ATP
assay for quantitative determination of mycobacteria were published
already in the 1970s [75].

Extraction of intracellular ATP. The extraction of ATP is an
important step for determination of intracellular ATP in cells of
mycobacteria. The extraction method must release the intracellular ATP
completely in one step and ensure minimal destruction of ATP during
extraction. It is rather difficult to extract ATP from mycobacteria
because the cell walls are very strong and “wax”-like. The
problem of ATP extraction from mycobacteria has received a considerable
amount of attention from many researchers.

Lysis of the cells with boiling Tris-EDTA buffer is the most common
method for extracting ATP from mycobacteria. The ATP is determined with
the bioluminescence method after cooling the lysate. The relationship
between ATP concentration and CFU/ml for serial dilutions of a sample
of Mycobacterium tuberculosis H37Rv culture was determined in
[76], and high correlation was observed
(r = 0.993). Nevertheless, when similar measurements were
conducted for eight different samples of the same strain taken
following different incubation time, the observed correlation
coefficient was slightly lower (r = 0.846). The authors
concluded that indeed the growth of the mycobacterial culture could be
monitored with the bioluminescence method. High bioluminescence signal
was observed already after 7 days of cell growth, while the signal was
lacking in sterile medium.

The authors of [77] compared three methods for ATP
extraction: 1) treatment with boiling Tris-EDTA buffer; 2) treatment
with hot chloroform; 3) treatment with butanol at room temperature.
Using fresh culture of the BCG variant Moreau strain of mycobacteria,
the ATP content was determined with the ATP bioluminescence method, and
number of bacteria from the mass of wet cells and concentration of
viable cells (CFU/ml) in the wet culture was determined by plating
serial dilutions and colony counting after 3-4 weeks of incubation. The
intracellular ATP extraction with butanol was found to be the most
effective. This simple and rapid technique was used by the authors in
further work. Correlation between the ATP concentration and CFU/ml with
correlation coefficient r = 0.99 was obtained for
suspensions containing (1-50)⋅107 CFU/ml. ATP content
per CFU/ml was compared for cells after 7, 9, and 12 days of growth. It
was found that the total ATP content in the culture and the number of
viable cells were the same for the seven- and nine-day cultures. The
total ATP content was approximately 10-fold lower for the
“aged” culture (12 and 16 days). However, the ATP content
of a single cell depended only slightly on the culture age and varied
in the range from 2.03 to 2.63 fg of ATP per CFU. This constitutes
approximately (3.7-4.8)⋅10–18 mol ATP per
cell. It must be noted that ATP content per cell can differ somewhat
for different strains of mycobacteria because it depends on the size of
the cell and its energy status. The storage of cells at different
temperatures as well as lyophilization can result in decrease in
intracellular ATP content.

The list of lytic agents was extended in [78]. The
following extractants were used: 1) boiling 0.1 M Tris-EDTA
buffer; 2) boiling 0.1 M Tris-EDTA buffer with addition of
detergent – 1, 2, or 4% dodecyltrimethylammonium bromide (DTAB);
3) 10% TCA; 4) 1 M NaOH; 5) DMSO. For extraction, an aliquot of
extractant (0.9 ml) was added to 0.1 ml of cell suspension,
mixed quickly, and either heated, when boiling buffer was used, or
incubated for a short time at room temperature. The resulting extracts
were stored for a few hours on ice, and then were frozen for long-term
storage. An internal standard was used during the ATP measurement (ATP
solution with concentration 100 nM). Comparison of extracts showed
that the NaOH released more than 90% of the ATP, but it could not be
used as it was hazardous. Other extractants released around 50% of ATP,
and only extraction with the use of boiling buffer with addition of 2%
DTAB resulted in release of 100% of the ATP. The results, however, cast
some doubt as the extraction method that uses TCA is considered as
classic for intracellular ATP extraction. Moreover, the correlation
between the ATP concentration and CFU for different extraction methods
is not presented in the work. The extraction method described in [78] was optimized by the authors in [79], and it was shown that the addition of 10%
cyclodextrin into the ATP-reagent decreased the inhibition of
luciferase by the DTAB.

The most significant work on the use of the bioluminescence ATP assay
for viability testing of the BCG vaccine is a study of Danish
scientists [80], where not only the method for
quantitative determination of the viability of the BCG vaccine based on
the amount of intracellular ATP was described, but the method was
thoroughly validated. They used industrial samples of lyophilized BCG
vaccine (strain 1331 from the Danish collection). Separate batches of
the vaccine were stored for various periods at different temperatures
to obtain samples with low (storage for 28 day at 37°C), medium
(storage at 2-8°C for 12-18 months), and high viability (storage at
2-8°C for 1-3 months). The lyophilized vaccine was resuspended in
diluted Sauton solution SSI, and apyrase solution was added to remove
extracellular ATP. Following short-term incubation, the intracellular
ATP was extracted using boiling Tris-EDTA buffer and the ATP
concentration was measured. Simultaneously, the number of CFU per vial
was determined by plating of serial dilutions, and the dependences of
the ATP content in the vial on the number of CFU per vial were
constructed.

The level of ATP in the vaccine analyzed immediately after
reconstruction was low. For example, the ATP content was practically
independent of the CFU value with CFU changing from 105 to
106 CFU/ml and was 2-4 ng per vial
(~10–18 mol ATP per CFU). This was explained by
the fact that the freezing and lyophilization represent a strong stress
for the cells, which results in a sharp drop of their metabolic
activity and, consequently, in decrease in the level of intracellular
ATP. For restoring metabolic activity of the cells, certain conditions
are required after rehydration of the cells. For this purpose, the
authors used preincubation for one day at 37°C in Dubos nutrient
medium containing Tween and albumin additives. This rich medium
restored metabolic activity of the cells, but the growth of the cells
was not yet initiated. Following incubation, the ATP content increased
5-10-fold in comparison with the non-incubated cells, while the number
of CFU remained the same. The average ATP content per CFU increased to
~10–17 mol. The conducted validation showed that
the method is sufficiently robust to accommodate minor changes in
incubation and ATP extraction conditions. Very high linear correlation
(r ≥ 0.99) was observed between the ATP content within the
range of 1-25 pmol ATP per vial, and the number of CFU per vial
for different samples of the same batch of the vaccine with high
viability. Good correlation (r = 0.928) between the ATP
contents in the range 8-45 ng per vial and the CFU numbers was
also observed for samples of vaccine batches with different viability.
This range of ATP content corresponds to the range of viable cells,
(0.7-7.6)⋅106 CFU per vial.

The Sauton solution SSI is used most often for regeneration of
lyophilized mycobacterial cells [81]. According to
[80], additional incubation in nutrient medium for
one day was required to restore cell viability completely and to obtain
good correlation between ATP content and CFU number. However, it was
shown in [82] that a 30-min incubation of the
lyophilized vaccine rehydrated in Sauton solution was sufficient for
restoration of viability of the lyophilized mycobacteria, and
satisfactory correlation between the ATP concentration and the CFU
numbers was observed. Hence, the one-day incubation in the nutrient
medium is not mandatory. This simplifies the process of the vaccine
pretreatment significantly.

Removal of extracellular ATP. A certain part of microbial cells
is destroyed during freezing and lyophilization. The intracellular ATP
is released into the solution in the process. Moreover, media used for
biomass production also can contain free, extracellular ATP. Hence, the
removal of extracellular ATP is an important step in the pretreatment
of reconstructed vaccine. In the case of vaccine, the best method for
removal of ATP from solution is treatment with apyrase enzyme solution
[83]. Usually the apyrase is isolated from potato
tubers [83]. The reaction catalyzed by apyrase is
described by the following scheme:

ATP → ADP + Pi; (1)

ADP → AMP + Pi. (2)

Neither ADP nor AMP is a substrate of firefly luciferase; hence,
decomposition of ATP to ADP and AMP results in 1000-fold decrease in
the emission. Two different apyrases were isolated from different
potato species: apyrase A, its activity in reaction (1) being ~10-fold
higher than in reaction (2); apyrase B, in contrast demonstrates
10-fold higher activity in reaction (2) rather than in reaction (1).

The use of apyrase A is necessary for the degradation of ATP, as it is
exactly this enzyme that ensures fast removal of free ATP from the
suspension of cells. It must be emphasized that apyrase does not
penetrate inside the cells, like other proteins, and therefore it does
not affect the intracellular ATP content. According to the literature,
10-min incubation of the cell suspension in dilute apyrase solution
decreases the concentration of the extracellular ATP by hundred-folds
[80]. In study [82], the
apyrase was added into the Sauton solution. The removal of
extracellular ATP occurred concurrently with the restoration of
metabolic activity of the cells.

Hence, the scheme for analysis of the rehydrated BCG vaccine is as
follows: in the first step the removal of extracellular ATP occurs, in
the second – extraction of intracellular ATP, and in the
third – determination of the intracellular ATP concentration
using the bioluminescence method (Fig. 4).

Fig. 4. Scheme of bioluminescence analysis of BCG
vaccine.

In conclusion, the literature data and data from our own studies on the
bioluminescence method for determination of cell viability and its
application in various areas of microbiology, sanitary control, and
ecology were analyzed in this review. The general characteristics of
the bioluminescence method for determination of extracellular and
intracellular ATP, methods for determination of ATP concentration, and
peculiarities of the determination of intracellular ATP in individual
and mixed cultures of microorganisms were considered, and the
efficiency of ATP extraction from cells using various lytic agents was
analyzed. The intracellular ATP content in cells of different types was
compared. The effect of different factors on the level of intracellular
and extracellular ATP was shown. Different examples of the application
of bioluminescence ATP assay for monitoring of food quality, hygienic
state of health care facilities and industrial clean rooms, for
investigation of different processes in cell systems (necrosis and
apoptosis of cells, peculiarities of cell growth and degradation,
effect of cellular stress on metabolic activity) were presented. The
advantages of the bioluminescence method for control of the specific
activity of vaccines based on live cells of lyophilized microorganisms
were shown.